GSM6685233: iMacrophage_oxENDseq; Mus musculus; OTHER
For END-seq, live cells were harvested and embedded into agarose plugs (Bio-Rad cat#1703591). Cells were lysed in agarose using proteinase K, follwed by Rnase treatment. DNA was later precipitated after plug melting and shearing. For S1-END-seq, cells were collected and embedded in 1% agarose plugs, lysed and digested with Proteinase K (1 hour at 50°C, followed by 7 hours at 37°C), washed with TE buffer, and then treated with RNAse A for 1 hour at 37°C. Plugs were then washed in EB and equilibrated in S1 nuclease buffer (40 mM sodium acetate pH 5.2, 300 mM NaCl, 2 mM ZnSO4) for 30 minutes. 1.8 U of S1 nuclease was added to 100 µL of S1 nuclease buffer per plug and incubated on ice for 15 minutes to allow for the enzyme to diffuse into the plug. The reaction mix was then placed at 37°C for 20 minutes before addition of EDTA (10 mM final concentration) to terminate the reaction. Finally, plugs were processed through the standard END-seq protocol Libraries were quantified using with the KAPA Library Quantification Kit (Kapa Biosciences) and sequenced in a NextSeq 550 system (Illumina, 75 bp single end reads).